Determination of Perfluoroalkyl and Polyfluoroalkyl ...

APPLICATION NOTE | www.LCTech-online.com As of: November 2019, Version: 1.2

APPLICATION NOTE | AN0033

Determination of Perfluoroalkyl and Polyfluoroalkyl Substances (PFAS) in Water

with FREESTYLE XANA and LC-MS/MS

Dr. Hans Rainer Wollseifen (MACHEREY-NAGEL) Sebastian Wierer (LCTech GmbH)

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Content

1. Introduction ................................................................................................................................................. 3

2. Method Development .............................................................................................................................. 4

Reagents ................................................................................................................................................ 4

Sample Preparation Standard/Blanks ........................................................................................ 4

Instrumentation .................................................................................................................................. 5

2.3.1 FREESTYLE XANA System ....................................................................................................... 5

Analytical Set-up ................................................................................................................................ 6

2.5.1 HPLC System and Settings ..................................................................................................... 6

2.5.2 Chromatographic Conditions ............................................................................................... 6

2.5.3 Eluent Exchange ........................................................................................................................ 6

2.5.5 Software Protocol ...................................................................................................................... 7

3. Results ............................................................................................................................................................. 8

Recovery Rates .................................................................................................................................... 8

Background Concentrations for PFAS ........................................................................................ 8

3.1 Chromatograms ............................................................................................................................... 10

4. Conclusions ............................................................................................................................................... 12

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1. Introduction

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are used in numerous industrial and consumer products because of their special chemical properties. The group of substances includes more than 3000 different compounds.

For more than 60 years, PFAS are used to provide everyday objects and materials with non-stick coatings. They make surfaces fat- and water-repellent and are extremely versatile. Not only functional clothing in the textile industry, but also technical devices and even pizza boxes or disposable coffee cups are often coated with PFAS. Other typical applications for PFAS would be fire-fighting foams, polishes, waxes and paint.

PFAS are anthropogenic and considered non-biodegradable. They get into the environment during their manufacturing process and also during their use and disposal. Therefore, PFAS can be found globally (even in polar regions) in the environment, in wildlife, in human tissues and blood. Sewage plants are mostly not able to purify waste water with PFAS, so it accumulates in the sewage sludge, that is often used in agriculture. The chemicals can reach the groundwater and get absorbed again through plants.

Humans mostly take in PFAS through food or contaminated drinking water. They can remain in the human body for years without being degraded. Animal studies showed, that PFAS can be carcinogenic, toxic for reproduction and weaken the immune system. It is still not clear, which effects PFAS have on the human body and what concentration must be reached to have an effect.

The topic of PFAS is present more than ever and will be even more relevant in the future as the European Comission has requested the EFSA to revaluate the risks of PFAS to human health. This could result in much lower detection limits and a wider range of analytes as known before.

The following application note shows how water samples can be prepared fully automated for LC-MS/MS analysis by applying SPE with the FREESTYLE XANA robotic system according to DIN 38407-42. By the application of fully automated parallel sample preparation, multiple samples can be processed at the same time. Thus, a high sample throughput at low demand of personnel resources is obtained.

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2. Method Development

Reagents 0.1 % ammonia in methanol

Acetonitrile (HPLC-grade)

Methanol (HPLC-grade)

Water (HPLC-grade)

1 % formic acid in acetone – acetonitrile (1:1, v/v)

5 mM ammonium acetate in water

5 mM ammonium acetate in methanol

Native PFAS precision and recovery standard solution (conc. 2.0 μg/mL in MeOH), Wellington Laboratories

Mass-labelled PFAS standard solution/mixture (conc. 2.0 μg/mL in MeOH), Wellington Laboratories

Sample Preparation Standard/Blanks Each standard was diluted to 200 ng/mL in methanol, as working solution. After that, 500 mL of water were spiked with 25 μL of each standard’s working solution. Accordingly, the sample has a concentration of 10 ng/L for each analyte, which is the lower application limit according to DIN 38407-42. The blanks were prepared as follows: All volumes of the solvents used in the application were put into a vial and evaporated to dryness , with a gentle stream of nitrogen. After that, the vial was rinsed with 1 mL acetonitrile. The solution again was concentrated to dryness under a gentle stream of nitrogen and redissolved with a final volume of 1 mL methanol/water (6:4, v/v).

The same procedure was applied with the methanol/water (6:4, v/v) solution.

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Instrumentation 2.3.1 FREESTYLE XANA System

The FREESTYLE XANA system consists of the xyz-robotic platform FREESTYLE BASIC and the SPE module which are modified and thus virtually free of fluorocarbon compounds for PFAS sample preparation. In the following the required items for a processing of 24 samples are listed together with their corresponding part numbers.

1. 1 x FREESTYLE BASIC, 6 Solvents P/N 12663-12-PFAS

2. 1 x FREESTYLE SPE Module P/N 12668-PFAS

3. 1 x FREESTYLE XANA P/N 14692-PFAS

4. 2 x Special Rack for up to 12 SPE Columns P/N 14047

5. 3 x Clamping Adapter for 3 mL SPE Cartridges P/N 14892

6. 1 x CHROMABOND HR-XAW, 3 mL, 60 mg P/N 17029

7. 2 x Reusable Polyethylene P/N 16970 Caps for SPE Cartridges

8. 2 x Reusable Needles, Stainless Steel P/N 13382

9. 1 x Frame 100 mm P/N 11915

10. 1 x Tray, 4 mL, 60 Positions P/N 11926

11. 1 x 4 mL Screw-Thread Vials P/N V0004

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Analytical Set-up 2.5.1 HPLC System and Settings

Agilent Infinity II 1290 (Modules G7116B, G7167B, G7120A)

API 5500 Triple Quad, Turbo Spray (ESI)

Scan type: SMRM

MRM detection window: 60 sec

Polarity: negative

Curtain gas: 30 psig

Ion spray voltage: -4500 V

Temperature: 400 °C

Gas 1 (nebulizer): 50 psig

Gas 2 (turbo gas): 60 psig

CAD gas: medium

2.5.2 Chromatographic Conditions

Column: EC 100/2 NUCLEOSHELL® Bluebird RP18 2.7 μm

Eluent A: 5 mM ammonium acetate in water

Eluent B: 5 mM ammonium acetate in methanol

Gradient: hold 20 % B for 1.0 min, in 8.0 min to 95 % B, hold 95 % B for 3.0 min, in 0.1 min to 20 % B, hold 20 % B for 2.9 min

Flow rate: 0.33 mL/min

Temperature: 40 °C

Injection volume: 10 μL (Concentration: 5 ng/mL in water/methanol (40/60, v/v))

2.5.3 Eluent Exchange

Sample concentration under a gentle stream of nitrogen to dryness

Rinsing with 1 mL acetonitrile

Sample concentration under a gentle stream of nitrogen to dryness

Fill up to a final volume of 1 mL with methanol/water (6:4, v/v)

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2.5.5 Software Protocol

In the following the FREESTYLE XANA method protocol is shown.

Figure 1: FREESTYLE XANA method protocol.

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3. Results

Recovery Rates Fourteen selected PFAS were processed on the FREESTYLE XANA system in order to see whether all compounds are recovered and do not adhere to the system.

Table 1: Recovery data of 14 selected PFAS compounds according to DIN 38407-42.

No Component Name Recovery [ %] STD [%] 1 Polyfluorobutanoic acid (PFBA) 80 3,8 2 Polyfluoropentanoic acid (PFPeA) 106 1,1 3 Polyfluorohexanoic acid (PFHxA) 101 1,3 4 Polyfluoroheptanoic acid (PFHpA) 106 1,5 5 Polyfluorooctanoic acid (PFOA) 107 1,6 6 Polyfluorononanoic acid (PFNA) 102 1,8 7 Polyfluorodecanoic acid (PFDA) 66 2,0 8 Polyfluorobutane sulfonate (PFBS) 102 1,4 9 Polyfluorohexane sulfonate (PFHxS) 107 2,1

10 Polyfluorooctane sulfonate (PFOS) 79 1,7 11 13C4 perfluorobutanoic acid (13C4 PFBA) 84 2,3 12 13C2 perfluorohexanoic acid (13C2 PFHxA) 108 1,6 13 13C4 perfluorooctanoic acid (13C4 PFOA) 113 1,3 14 13C4 perfluorooctanesulfonic acid (13C4 PFOS) 85 1,8

Background Concentrations for PFAS One of the key challenges in analysing PFAS is to keep the background of analytes as low as possible. Unfortunately, PFAS are widely spread all over the globe and will thus be found ubiquitously. Furthermore, any fluorocarbon materials, commonly used in laboratory systems due to their unique chemical properties, are prone to a release of small amounts of PFAS that may significantly increase background levels.

Therefore, any fluorocarbon materials of the FREESTYLE SPE as well as the XANA system were consequently removed and replaced by polyethylene or polypropylene, respectively.

In order to prove the effectiveness of this measure, PFAS background levels of neat solvents and pure water samples were compared.

From the Table 2 shown below, it is evident that any analyte concentration measured is already found in the neat solvents and does not originate from the system!

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For the determination of the background concentration of the solvents and solutions, the processing was as described in 2.2.

For the determination of the background concentration of the FREESTYLE XANA system, the application was processed with 500 mL of unspiked water and the same solvents, that were processed as described in 2.2.

Table 2: Comparison of PFAS background concentrations of neat solvent and water samples processed with the FREESTYLE XANA system

Component Name

Background FREESTYLE

[ng/mL]

Background Solvent [ng/mL]

PFBA 0 0 PFPeA 0,09 0,06 PFHxA 0,02 0,04 PFHpA 0,02 0,02 PFOA 0,05 0,05 PFDA 0,01 0,01 PFNA 0,01 0,01 PFBS 0 0

PFHxS 0 0 PFOS 0,01 0,01 PFDS 0,01 0,01

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3.1 Chromatograms

Figure 2: Chromatogram of spiked sample over FREESTYLE XANA

Figure 3: Chromatogram of blank solvent

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Figure 4: Chromatogram of blank sample over FREESTYLE XANA

Table 3: LC-MS/MS parameters

No Component

Name Retention Time

(Min.)

MRM-Transition

Q1 Q2 Q3

1 PFBA 2,57 212,9 168,8 -

2 PFPeA 4,68 262,9 219 -

3 PFHxA 5,88 312,9 268,7 119

4 PFHpA 6,65 362,9 318,6 168,8

5 PFOA 7,23 412,9 368,8 168,8

6 PFNA 7,7 462,9 418,9 218,9

7 PFDA 8,1 512,8 468,9 218,8

14 PFBS 5,02 298,9 79,9 98,9

15 PFHxS 6,69 398,9 79,8 98,8

17 PFOS 7,69 498,9 79,9 98,9

25 M4-PFBA 2,56 216,9 171,9 -

26 M4-PFOA 7,23 416,8 371,8 171,9

27 M2-PFHxA 5,88 314,9 269,9 120

30 M4-PFOS 7,68 502,8 79,9 98,9

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4. Conclusions

In the application note, PFAS can be processed fully automated according to DIN 38407-42. Due to the specifically designed PFAS system, which is virtually free of fluorocarbon compounds, any blank values stemming from the system are minimised. This was shown by PFAS background measurements of the instrument that were equal to the values obtained with neat solvent. Furthermore, the analytical targets of the DIN method were found with good recoveries and very low standard variations because of the reliable and robust automation. As the FREESTYLE SPE module is an integral part of the FREESTYLE XANA, it is evident that a processing on the SPE system only is possible as well.

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Kontakt

LCTech GmbH Daimlerstraße 4 84419 Obertaufkirchen Deutschland Tel.: +49 8082 2717-0 Fax: +49 8082 2717-100 E-Mail: [email protected] www.LCTech.de www.LCTech-online.com

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